Gene expression reveals evidence for EGFR-dependent proximal-distal limb patterning in a myriapod

DOI: 10.1111/ede.12222 RESEARCH PAPER Gene expression reveals evidence for EGFR-dependent proximal-distal limb patterning in a myriapod Ralf Janssen Department of Earth Sciences, Uppsala University, Uppsala, Sweden Correspondence Ralf Janssen, Department of Earth Sciences, Uppsala University, Palaeobiology, Villavägen 16, 75236 Uppsala, Sweden Email: ralf.janssen@geo.uu.se Funding information Vetenskapsradet, Grant number: 621-2011- 4703 Evolution of segmented limbs is one of the key innovations of Arthropoda, allowing development of functionally specific specialized head and trunk appendages, a major factor behind their unmatched evolutionary success. Proximodistal limb patterning is controlled by two regulatory networks in the vinegar fly Drosophila melanogaster, and other insects. The first is represented by the function of the morphogens Wingless (Wg) and Decapentaplegic (Dpp); the second by the EGFR-signaling cascade. While the role of Wg and Dpp has been studied in a wide range of arthropods representing all main branches, that is, Pancrustacea (= Hexapoda + Crustacea), Myriapoda and Chelicerata, investigation of the potential role of EGFR-signaling is restricted to insects (Hexapoda). Gene expression analysis of Egfr, its potential ligands, and putative downstream factors in the pill millipede Glomeris marginata (Myriapoda: Diplopoda), reveals that in at least mandibulate arthropods EGFR-signaling is likely a conserved regulatory mechanism in proximodistal limb patterning. 1 INTRODUCTION Arthropoda comprises the largest animal phylum on the planet with estimated more than five million species and a countless number of individuals (e.g., Odegaard, 2000 and references therein). Fundamentally, the arthropod body is composed of serially homologous units arranged along the anterior posterior body axis; these segments, each equipped with their own set of limbs, are among the key features that may have facilitated the phylum s evolutionary success. Another feature, unique to the arthropods, is their segmented limbs. Like the compartmentalization of the body, modular organization of the limbs likely permitted arthropods to evolve into a myriad of forms, each perfectly suited to new and changing environments. This adaptability is why arthropods can be found in virtually every habitat, a success story that dates back to at least the early Cambrian some 520 Mya (e.g., Budd & Jensen, 2000; Edgecombe, 2010). The jointed appendages of arthropods are believed to have evolved from simpler, tube-like forms, as represented by the limbs of onychophorans and tardigrades (reviewed in Shubin, Tabin, & Carroll, 1997). A current hypothesis suggests that the ancestral arthropod limb was bipartite, with a proximal segment that represented the main part of the modern arthropod limb, and a second region that represented the distal region including the tip of the modern arthropod limb (Casares & Mann, 2001). In the vinegar fly Drosophila melanogaster, the main arthropod model organism, the proximodistal (PD) axis of the limbs is patterned by the morphogens Wingless (Wg) and This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 2017 The Authors. Evolution and Development Published by Wiley Periodicals, Inc. 124 wileyonlinelibrary.com/journal/ede Evolution & Development. 2017;19:124 135.

JANSSEN 125 Decapentaplegic (Dpp) (Campbell & Tomlinson, 1998; Campbell, Weaver, & Tomlinson, 1993; Diaz-Benjumea, Cohen, & Cohen, 1994), which activate genes along the PD axis. It was found that activation of PD-axis patterning-genes is also under control of epidermal growth factor-related (EGF) ligands such as the neuregulin Vein (Vn) (and/or the TGF-alpha family members Keren [Krn] and Spitz [Spi]) from a source in the tip of the limbs that results, via binding to the EGF-receptor (EGFR), in a gradient of EGFR-Ras signaling declining distal to proximal (Campbell, 2002; Galindo, Bishop, Greig, & Couso, 2002). As a result, genes such as aristaless (al), clawless (cll), and Lim1 become expressed in the very tip of the limb, while Bar (B) and rotund (rn) are activated in a nearby sub-terminal ring (Campbell, 2002; Campbell et al., 1993, Galindo et al., 2002; Kojima, Sato, & Saigo, 2000; Kojima, Tsuji, & Saigo, 2005; Pueyo & Couso, 2004; Pueyo, Galindo, Bishop, & Couso, 2000). The roles of Wg and Dpp in early PD-axis patterning are widely conserved in arthropods, for example in the red flour beetle Tribolium castaneum and the Tiger wandering spider Cupiennius salei (Beermann, Prühs, Lutz, & Schröder, 2011; Ober & Jockusch, 2006; Prpic, Janssen, Wigand, Klingler, & Damen, 2003). However, distal morphogen-signaling appears to be somewhat different in Drosophila compared with other arthropods and onychophorans (Akiyama-Oda & Oda, 2003; Grossmann & Prpic, 2012; Janssen, Jorgensen, Prpic, & Budd, 2015; Jockusch, Nulsen, Newfeld, & Nagy, 2000; Prpic et al., 2003; Sanchez-Salazar et al., 1996; Smith, Angelini, Gaudio, & Jockusch, 2014), a circumstance that could be explained by the so-called topology model (Angelini & Kaufman, 2005; Prpic et al., 2003). This concept was introduced on theoretical grounds by Almirantis and Papageorgiou (1999) and discussed on experimental evidence by Prpic et al. (2003) explaining the differences seen in morphogen-expression in essentially two-dimensional structures, the Drosophila imaginal discs, compared to the situation in three-dimensional systems as seen in the outgrowing limbs of spiders (and other arthropods with a typical developmental mode) (Prpic et al. 2003). The term topology model was first used by Angelini and Kaufman (2005) where they discussed the findings of Prpic et al. (2003) in their paper. A role for EGFR-signaling in PD-axis patterning has been shown for the beetle Tribolium. However, in this species EGFR-signaling is also responsible for correct medial limb development (Grossmann & Prpic, 2012; Smith et al., 2014). In the two-spotted cricket Gryllus bimaculatus, correct regeneration of amputated nymphal legs fails in the absence/downregulation of EGFR-signaling (Nakamura, Mito, Miyawaki, Ohuchi, & Noji, 2008) supporting the view that even in hemimetabolous insects EGFR-signaling could represent a crucial component of limb patterning. Although data are convincing that EGFR-signaling is a vital component of PD-axis patterning in insect limbs (as exemplified by studies on Drosophila, Tribolium, and Gryllus), it remains unclear if this function is restricted to insects, or whether it is a deeper component of the ancestral arthropod limb-patterning network. To shed further light onto this topic, components of EGFR-signaling such as Egfr, its potential ligand-encoding genes, and a number of potential downstream factors, were studied here in the pill millipede Glomeris marginata. The new data suggest that EGFR-signaling is involved in myriapod PD-limb patterning, implying that this gene regulatory network was used for limb patterning in the last common ancestor of Mandibulata (Pancrustacea [= Hexapoda + Crustacea] + Myriapoda). The new data also allow for the tracing of evolutionary changes and the origin of distal limb patterning genes in (pan)arthropods. In order to obtain a better basis for comparison, two hitherto uninvestigated limbpatterning genes of the onychophoran Euperipatoides kanangrensis were also investigated. 2 METHODS 2.1 Embryo collection, fixation, and staging Embryos of G. marginata and E. kanangrensis were collected, fixed, and staged as described in Janssen, Prpic, and Damen (2004) and Janssen and Budd (2013), respectively. 2.2 Gene cloning and sequence analysis Total RNA was isolated from embryos of different stages using TRIZOL (Invitrogen). This RNA was reverse transcribed into cdna (SuperscriptII first strand synthesis system for RT-PCR, Invitrogen). Gene fragments were isolated by means of PCR with gene specific primers (based on a sequenced embryonic transcriptome). In all cases a first PCR was followed by a second (nested) PCR. Sequences of all fragments were determined from both strands on a 3100 automated sequencer, Terminator Cycle Sequencing Kit; Perkin-Elmer Applied Biosystems, Foster City, CA, USA) using Big Dye dye-terminators version 3.1 (Big Dye Terminator Cycle Sequencing Kit; Perkin-Elmer Applied Biosystems), and cloned into pcr-ii vectors (TA Cloning Kit Dual Promoter, Invitrogen, Carlsbad, CA, USA). 2.3 Sequence analysis Conserved domains of G. marginata Clawless (Cll), Aristaless (Al), Lim1, Rotund (Rn), and Zinc finger homeodomain 2 (Zfh2) were aligned by hand and compared with their arthropod orthologs and conserved domains of closely related genes (Oliveira et al., 2014). Putative sequences of Glomeris

126 JANSSEN disconnected (Gm-disco), Bar (Gm-B), vein (Gm-vn), keren (Gm-krn), Epidermal growth factor receptor (Gm-Egfr), and Euperipatoides disconnected (Ek-disco) andbar (Ek-B) were identified in the sequenced embryonic transcriptomes of Glomeris and Euperipatoides. Gene-identity was then validated by reciprocal BLAST searches of the sequences against generic arthropod databases. Sequence information is available under accession numbers LT716782 (Gm-Egfr), LT716783 (Gm-vn), LT716784 (Gm-krn), LT716785 (Gmcll), LT716786 (Gm-al), LT716787 (Gm-B), LT716788 (Gmrn), LT716789 (Gm-disco), LT716790 (Gm-Lim1), LT716791 (Gm-zfh2), LT716792 (Ek-disco), and LT716793 (Ek-B). 2.4 Whole mount in situ hybridization, nuclear staining, and dissection and mounting of appendages In situ hybridization was performed as described in Janssen et al. (2015). Nucleic staining was done by incubation of the embryos in 1 mg/ml of the fluorescent dye 4-6-diamidin-2- phenylindol (DAPI) in phosphate-buffered saline with 0.1% Tween-20 (PBST) for 20 30 min. Prior to dissection of appendages, embryos were incubated in glycerol (10 min incubation in 25% glycerol in PBST, followed by the same incubation in 50%, 75%, and 87% of glycerol). Appendages were dissected using a fine tungsten needle taken from an old light-bulb and sharpened in the flame of a Bunsen burner. 2.5 Data documentation Embryos were analyzed under a Leica dissection microscope equipped with a Leica DC100 digital camera. The image processing software Adobe Photoshop CS2 (Version 9.0.1 for Apple Macintosh) was used for linear corrections of brightness, contrast, and color values in all images. 3 RESULTS 3.1 Expression patterns of Glomeris Epidermal growth factor receptor (Egfr) and its potential ligands 3.1.1 Epidermal growth factor receptor (Gm-Egfr) First expression is seen in the mouth primordium (not shown). At stage 1.2, additional expression appears in the outgrowing dorsal segmental units (Figure 1a). Most probably this expression is associated with tergite boundary formation (cf. Janssen et al., 2004, Janssen, Budd, Damen, & Prpic, 2008). At stage 3, expression appears along the ventral midline (Figure 1b). From stage 4 onwards, Gm-Egfr is ubiquitously expressed (Figures 1c,d and 2), however, expression is still at higher levels in the previously described structures. At stage 4.1, expression in the ventral midline is restricted to nascent (posterior) segments; in more anterior segments, Egfr is expressed in segmental patches in the ventral nervous system and in longitudinal stripes ventral to the base of the limbs (Figure 1d). From stage 5 onwards the complete ventral nervous system and the brain strongly express Egfr (Figure 2a,b). Expression is enhanced in the distal tips of the antennae (Figure 2b). 3.1.2 vein (Gm-vn) First expression of Gm-vn is detectable at early developmental stages when it is in the ocular region and ubiquitously in the primordia of the trunk segments and the segment addition zone (SAZ) (Figure 1e). Later, expression occurs in a saltand-pepper pattern in the complete germ band (Figures 1f h and 2g). Strong expression occurs in the tips of the walking limbs (Figures 1f h and 2k,l). At stage 4, expression appears in the ventral nervous system (Figures 1h and 2g). The labrum expresses Gm-vn in all dorsal tissue (Figure 2h). In the antennae, expression is restricted to a small domain near the distal tips (Figure 2h). The mandibles express Gm-vn along their dorsal side and in form of a distal and ventral patch (Figure 2i). Expression in the maxillae is restricted to medial and dorsal tissue (Figure 2j). At early stages of walking limb development vn is expressed in the complete limb buds (Figure 2l), but the late walking limbs only express Gm-vn in a small domain in their distal tips and in a broad medial and proximal domain (Figure 2k). Proximally in the legs, however, this expression is restricted to anterior tissue; ventral, dorsal and posterior tissue is free from expression. 3.1.3 keren (Gm-krn) The earliest expression of Gm-krn is visible at approximately stage 5 when it is in the labrum, the antennae, and the maxillae, but not the mandibles and the walking limbs (not shown). Shortly later, expression also appears in the mandibles and the walking limbs (Figure 2m r). At this point, small domains in the ventral nervous system of the nascent trunk segment T6 and the anterior of the anal valves express Gm-krn (Figure 2m). In the head, two small domains appear in the ocular region, suggesting a conserved role in eye development (Figure 2n) (Brown, Kerr, & Freeman, 2007). Two dots of expression also appear in the labrum (Figure 2n). Four distinct dots of expression appear in the tip of the antennae; most probably this expression is in the developing sensory cones (sensory structures; cf. expression of other genes in this specific pattern [Prpic & Tautz, 2003, Prpic, Janssen, Damen, & Tautz, 2005]). The mandibles express Gm-krn between the inner and outer distal lobes and in a diffuse internal pattern (Figure 2o). Within the maxillae two domains of expression appear, one at the ventral and anterior base, and one weaker in the center

JANSSEN 127 FIGURE 1 Expression of Egfr and vn. Expression of Egfr (a d) and vn (e h). In all panels, anterior is to the left, ventral view. (a d) Asterisks mark expression in the dorsal segmental units. The arrows in c and d mark expression along the ventral midline (Note the changed pattern in more anterior segments). The black arrowhead in d marks expression ventral to the base of the limbs. The white arrowhead in d indicates expression in the ventral nervous system. Arrowheads in f h mark expression in the tip of the first walking limb. The arrow in h points to expression in the ventral nervous system. oc, ocular region; s, stomodaeum; st, developmental stage (after Janssen et al., 2004) (Figure 2p,q). A distal and ventral domain of expression appears later in the walking limbs (Figure 2r). 3.2 Expression patterns of potential downstream factors of EGFR-signaling 3.2.1 aristaless (Gm-al) Expression of Gm-al appears first at stage 1.1 in the primordia of the mandibles and the maxillae (Figure S1a,a ). Somewhat later it appears in the antennal primordium (Figure 3a) and in the first three walking limbs (Figure 3b). Expression in the antennae is transient, but expression in the mandibles and maxillae becomes stronger (Figure 3c). Additional expression appears in patches dorsal to the walking limbs (Figure 3c,d). Weak expression is in the medial part of the antennae and in the center of the labrum (Figure 4a). The complete mandibles express Gm-al (Figure 4b), while expression in the maxillae and the walking limbs is restricted to dorsal tissue (Figure 4c, d). In the walking limbs, expression is stronger in the tip and in proximal tissue (Figure 4d). Younger walking limbs, as represented by the walking limb on the fourth trunk segment (T4) only weakly express al (Figure 4e). 3.2.2 Bar (Gm-B) Gm-Bar is first expressed in broad domains covering the primordia of the mandibles and the maxillae (Figure 3e). Somewhat later, when the limb buds grow out, expression appears in the antennal anlagen and in two distinct dots lateral to the stomodaeum (Figure 3f). Gm-B is expressed in the center of the labrum and in form of a sub-terminal ring in the antennae and the walking limbs (Figures 3g and 4f,i); but note that this ring is not present in young limb buds (Figure 4j). In the mandibles, expression is restricted to dorsal and proximal tissue (Figure 4g). This expression appears at stage 6. In the maxillae, Gm-B is expressed in three distinct ventral domains (Figures 3g and 4h). This domain resembles expression of Distal-less (Dll), wg/wnt1, Wnt6, Wnt9, and Wnt11, and is likely corresponding to the developing sensory maxillary structures (Janssen & Posnien, 2014; Prpic, 2004; Prpic & Tautz, 2003). 3.2.3 clawless (Gm-cll) Expression of Gm-cll is first detectable at stage 2 in the tips of the developing antennal buds (Figure S1b,b ). At stage 3, expression appears in the dorsal germ band (the later heart) and the anal valves (Figure 3i). At stage 4, expression appears in the tips of the first three walking limbs and the anal valves (Figure 3j,k). At stage 6.1, Gm-cll is also expressed in the tips of the fourth walking limb (Figures 3l and 4o). Weak expression is detectable in the center of the mandibles from stage 5 onwards. Maxillae and the labrum never express Gm-cll (Figure 4k m). 3.2.4 Lim1 (Gm-Lim1) Lim1 is ubiquitously expressed at low levels. Enhanced expression is first seen anterior to the base of the antennae,

128 JANSSEN FIGURE 2 Expression of Egfr, vn, and krn. Expression of Egfr (a f), vn (g l) and krn (m r). Asterisks in a mark expression in the forming tergite boundaries. The arrowhead in g indicates expression in the ventral nervous system (VNS). Red arrowhead in panel k marks expression in the tip of the walking limbs. Arrowhead and arrow in panel m mark expression in the VNS and the anal valves respectively. Arrow in panel n indicates expression in the antennal cones. Red arrowhead in panel r marks expression near the tips of the walking limb. an, antenna; av, anal valve; br, brain; lr, labrum; md, mandible; mx, maxilla; oc, ocular region; pmx, postmaxillary segment (limbless); st, developmental stage (after Janssen et al., 2004); T1, first walking limb; T4, fourth walking limb; vns, ventral nervous system the mandibles, and the maxillae (Figure 3m), and later these structures continue to express Lim1 at high levels (Figure 3n p). Within the antennae, Lim1 is expressed in three distinct domains, a proximal, a medial and a sub-terminal domain (Figure 4u). The complete mandibles express Lim1 at a high level (Figure 4v), but in the maxillae strong expression is restricted to the distal region (Figure 4w). Expression in the walking limbs is ubiquitous (Figure 4x,y). 3.2.5 rotund (Gm-rn) Gm-rn is not expressed in young embryos. At stage 3, expression is in all outgrowing appendages, except the labrum (Figure 3q). Weak expression is in the dorsal segmental units (Figure 3q). At later stages the overall expression of Gm-rn becomes stronger and additional expression appears in a segmental pattern in the ventral nervous system (Figure 3r,s). Expression in the appendages is ubiquitous, but stronger in mandibles and maxillae (Figure 4z d ). Within the labrum and the antennae regions of slightly stronger expression can be detected. In the labrum, this region lies dorsally in the tip and in the antennae it lies ventrally in the tips (Figure 4z). 3.3 Expression of additional distal limb patterning genes 3.3.1 disconnected (Gm-disco) At early developmental stages, Gm-disco is expressed in a broad anterior domain representing the region of the future

JANSSEN 129 FIGURE 3 Expression of al, B, cll, Lim1, rn, disco, and zfh2. In all panels, anterior is to the left, ventral view; except for panel t (lateral view). Developmental stage is indicated. (a d) Expression of al. (e h) Expression of B. (i l) Expression of cll. (m p) Expression of Lim1. (q t) Expression of rn. (u x) Expression of Disco. (y z ) Expression of zfh2. Arrowheads in panels c,d mark expression in dorsal segmental tissue. The arrowheads in f/g point to expression lateral to stomodaeum and labrum. Arrowhead in l marks expression in T4. Asterisks in p and s mark dorsal segmental expression. an, antenna; av, anal valve; br, brain; h, heart; lr, labrum; md, mandible; mx, maxilla; oc, ocular region; T1, first walking limb; T4, fourth walking limb; vns, ventral nervous system

130 JANSSEN FIGURE 4 Expression in head appendages and walking limbs. (a e) Expression of al. (f j) Expression of B. (k o) Expression of cll. (p t) Expression of disco. (z y) Expression of Lim1. (z d ) Expression of rn. (e i ) Expression of zhf2. Arrowheads in panels e and h mark ring of expression in antennae and walking limbs respectively. an, antenna; lr, labrum; oc, ocular region labrum, and in the primordia of the anterior appendages, as well as the SAZ (Figure S1c). Co-expression of Gm-disco with either Glomeris engrailed (Gm-en) (Janssen et al., 2004) (Figure S1d) or collier (Gm-col) (Janssen, Damen, & Budd, 2011) (Figure S1e) reveals that the early segmental expression corresponds to the limb primordia of the antennae, the mandibles, and the maxillae. At later stages, expression is in the primordia of all limbs including the tissue of the limb-less postmaxillary segment; here, however, expression is weaker than in the limb-bearing segments (Figure 3u). Expression in the anal valves persists throughout development (Figure 3u x). Expression in the labrum, the antennae and the maxillae is in all tissue except for the distal tip, which is free from expression (Figure 4p t). In the mandibles, the internal mandibular lobe expresses Gm-disco. The walking legs first express disco ubiquitously, but later during development expression disappears from the distal region (cf. Figure 4s,t). 3.3.2 zinc finger homeodomain 2 (Gm-zfh2) The earliest differential expression of Gm-zfh2 is in the head lobes and the ventral nervous system. At this stage (stage 0.4), the complete ectoderm (including extraembryonic tissue) expresses Gm-zfh2 (Figure S1f). Somewhat later, only the central nervous system (CNS) expresses Gm-zfh2 (Figure 3y). The SAZ and the last newly formed posterior segment(s) do not express Gm-zfh2 (Figure 3y). The strong expression in the CNS remains throughout embryogenesis until at least stage 6.1 (Figure 3y z and not shown). Gm-zfh2 is also expressed in dorsal segmental tissue and the limbs, but at lower level than in the CNS (Figure 3z ). A clear ring of expression is located close to the tip of the antennae (Figure 4e). Enhanced expression in the mandibles and the maxillae is restricted to ventral tissue (Figure 4f,g). In late-stage walking limbs the situation is comparable to that in the antennae (Figure 4h), but

JANSSEN 131 in younger walking limbs there is no sub-terminal ring (Figure 4i). 3.4 Expression of onychophoran Bar (Ek-B) and disconnected (Ek-Disco) Ek-B is expressed in the tips of the frontal appendages, the slime papillae and the walking limbs (Figure S2a). Later, expression appears as dots in the anterior of the head lobes, and additional cells in the frontal appendage and along the body axis express B (Figure S2b,c). The expression of Euperipatoides B is likely associated with the development of neuronal sensory structures. Ek-disco is first expressed in all outgrowing limb-buds (Figure S2d,e). Later it is also expressed in the head anterior to the position of the mouth (Figure S2f,k). Expression in the limbs transforms into a domain along the posterior of the frontal appendages, the complete jaws, and a median/distal region of the walking limbs and the slime papillae (Figure S2g k). In both the slime papillae and walking limbs, expression is strongest in the middle forming a leg gapgene like expression domain (cf. expression of dachshund [Janssen, Eriksson, Budd, Akam, & Prpic, 2010]). The distal region of these limbs express disco at a lower level. A close inspection of the jaws reveals that even here, expression is somewhat weaker in the tips (Figure S2i,k). 4 DISCUSSION 4.1 Prolog: The gnathobasic nature of the arthropod mandible and maxilla It is widely accepted that the walking limbs of arthropods, onychophorans, tardigrades and the extinct lobopodians represent the most ancestral type of limbs (e.g., Angelini, Smith, & Jockusch, 2012; Eriksson, Tait, Budd, Janssen, & Akam, 2010). This assumption is supported by the fossil record that presents numerous panarthropodian fossils with a series of identical limbs along the body that are used for locomotion, and a minor number of specialized anterior appendages (e.g., Boxshall, 2004; Budd, 1996; Ramsköld, Chen, Edgecombe, & Zhou, 1997; Vannier & Chen, 2000; Whittington, 1981). While the walking limbs and the antennae of mandibulate arthropods clearly possess well-developed telopodites (reviewed in Boxshall, 2004, 2013), the gnathal appendages (mandibles and maxillae) are likely of gnathobasic nature, and thus lack distal components of the typical limb. Indeed, for Glomeris it has been shown that typical distal limb patterning genes such as Distal-less are not expressed in the gnathal appendages (Popadic, Panganiban, Rusch, Shear, & Kaufman, 1998; Prpic, 2004; Prpic & Tautz, 2003; Prpic et al., 2005; Scholtz, Mittmann, & Gerberding, 1998). This study corroborates these earlier findings in that several of the limb-tip patterning genes are not expressed in the mandibles and maxillae, or are expressed in diverged patterns, while more conserved patterns can be seen in both developing antennae and walking limbs (cf. expression patterns summarized in Figures 4 and 5). Further analysis therefore focuses on possibly conserved expression patterns in distal limb development in the walking limbs and antennae. 4.2 Evidence for EGFR-signaling in myriapod limbs The epidermal growth factor receptor (EGFR) plays a pivotal role in EGFR-signaling. In Glomeris, Egfr is ubiquitously expressed and thus the prerequisite for EGFR-signaling is given in all tissue, including the limbs. However, EGFRsignaling requires the binding of a ligand. In Drosophila, at least four such ligands have been identified, that is, Vein (Vn) (Schnepp, Grumbling, Donaldson, & Simcox, 1996), Spitz (Spi) (Rutledge, Zhang, Bier, Jan, & Perrimon, 1992), Keren (Krn) (Reich & Shilo, 2002), and Gurken (Grk) (Neuman- Silberberg & Schupbach, 1993). One level of specificity of EGFR-signaling is provided by specific binding/presence of these ligands; for example, Gurken is only expressed in the Drosophila oocyte (Neuman-Silberberg & Schupbach, 1993). In Drosophila, Vn appears to be the specific limb-patterning ligand (Galindo, Bishop, & Couso, 2005), whereas in Tribolium, Spi/Krn seems to fulfill a similar function (note that the single TGF-alpha type gene in Tribolium is alternatively called spitz or keren) (Angelini, Kikuchi, & Jockusch, 2009; Grossmann & Prpic, 2012). Interaction of EGFR and its potential ligands during limb development thus appears to be rather flexible. It was therefore necessary to study both potential EGFR-ligands identified in Glomeris, that is Gm-krn, and Gm-vn. Gene expression analysis revealed that vn is expressed in a specific pattern that implies a role in PD limb-patterning, that is, expression in the tip of the outgrowing legs. This expression suggests that vn mrna may act as a source of Vn protein in the distal tip of the legs, which could form a distal-to-proximal protein gradient comparable to the situation for Drosophila (Campbell, 2002; Galindo et al., 2002), and by the proposed function of Spi in Tribolium (Grossmann & Prpic, 2012). The expression profile of Glomeris krn, although expressed in the appendages, does not suggest a role in PD limb patterning. It appears probable that the expression of krn is associated with formation of sensory structures, such as the antennal cones. Overall, the situation is thus similar in Drosophila and Glomeris in that Vn may be the limb-specific EGFR-ligand. To further analyze PD limb-patterning in Glomeris, and to provide additional evidence for the involvement of an EGFRsignaling gradient in PD limb-patterning, the expression of

132 JANSSEN FIGURE 5 Conserved and diverged expression of limb-patterning genes in panarthropods. This schematic drawing summarizes known mrna expression patterns in the fly D. melanogaster, the beetle T. castaneum, the pill millipede G. marginata, and the onychophoran E. kanangrensis. Note that the expression of Euperipatoides Egfr is ubiquitous (unpublished data). Expression is in red. Light red indicates weaker expression. Question marks indicate that information about mrna expression is not available. Note that the drawing is simplified and does not show all aspects of expression in the walking limbs at all developmental stages. The tree represents a simplified panarthropod phylogeny showing the most-likely relationship of the main arthropod taxa and their close relatives, the onychophorans and the tardigrades (based on Campbell et al., 2011). The double-arrow indicates the still unresolved relationship of onychophorans and tardigrades relative to Arthropoda downstream factors of EGFR-signaling have been investigated (discussed below). 4.3 Downstream of EGFR-signaling In Drosophila, high levels of EGFR-signaling activate the expression of al, cll, and Lim1 in the very center of the leg imaginal disc (Campbell, 2002, 2005; Galindo et al., 2002, 2005). Lower levels of EGFR-signaling are needed to activate the expression of B, which is therefore expressed in a ring proximal to the expression of al, cll, and Lim1. Restricted expression of al and cll to the tip of the limbs is observed in Tribolium (Beermann & Schröder, 2004; Grossmann & Prpic, 2012), in (investigated for al) Gryllus (Miyawaki et al., 2002), and indeed the myriapod Glomeris (this study) (Figures 4 and 5). Most strikingly, the very specific expression of B in the form of a ring proximal to the expression of al/cll is conserved in Glomeris. A similar pattern is also present in the onychophoran (although note that this pattern is likely associated with the formation of sensory/neuronal structures), (Figures 4, 5, and S2). The expression profiles seen in arthropods suggest a conserved distal limb-patterning

JANSSEN 133 network, possibly under control of EGFR-signaling, as in the case of Drosophila. Nonetheless, other distal factors are expressed very differently in Glomeris, such as Lim1 and rn both of which are expressed in the complete legs, as opposed to the tip and a ring respectively (Pueyo et al., 2000; St Pierre, Galindo, Couso, & Thor, 2002). 4.4 Tracing the origin of distal limb patterning genes in Panarthropoda The expression of al, B, and cll in the tips of the walking limbs of Drosophila, other insects and the myriapod Glomeris is comparable, and thus probably conserved during at least mandibulate evolution. Data from the onychophoran Euperipatoides (Oliveira et al., 2014, Figure S2) suggests that the patterns of al, B, cll, disco, and rn may originate from the ancestor of arthropods + onychophorans, although possibly conserved patterns of rn are limited to Tribolium and Euperipatoides, and the patterns of disco and B are somewhat different in onychophorans and arthropods (summarized in Figure 5). The expression pattern of Lim1 in Drosophila and the onychophoran is comparable. However, for Lim1 data are scarce and the pattern in Glomeris is different from that seen in Drosophila and Euperipatoides. Our knowledge on the expression of the investigated limb patterning genes suffers from a general paucity of data from crustaceans and chelicerates, as well as a lack of data from insects other than Drosophila, including from Tribolium (summarized in Figure 5). Comparative data from a tardigrade would be needed to further investigate the origin and ancestral nature of their expression, but unfortunately such data are not yet available. The majority of the investigated Drosophila distal limb-patterning genes downstream of EGFR-signaling, however, are expressed in similar patterns, suggesting the possibility of an ancestral underlying gene regulatory network, which is likely represented by EGFR-signaling. 4.5 Addendum: Two more conserved distal limb patterning genes Two additional limb-patterning genes, zinc finger homeodomain 2 (zfh2) and disconnected (disco) have been investigated in this study, although they do not represent known downstream targets of EGFR-signaling. Both genes are expressed in complex and conserved, or at least similar (for disco) patterns in the limbs of arthropods and the onychophoran Euperipatoides (Oliveira et al., 2014) (summarized in Figure 5). Although the regulation of zfh2 in insects is unclear, it has been identified as a limb-patterning gene with a specific function in distal limb development (Guarner et al., 2014; Kittelmann et al., 2009; Lai, Fortini, & Rubin, 1991); a function that may date back to the last common ancestor of arthropods and onychophorans. In Drosophila, the two disco genes interact with the limbpatterning master-gene Distal-less (Dll) and are involved in the specification of ventral versus dorsal appendages (Dey, Zhao, Popo-Ola, & Campos, 2009; Patel et al., 2007). Interestingly, disco genes are strongly expressed in the tips of legs and antennae in Drosophila (Cohen, Wimmer, & Cohen, 1991; Lee et al., 1991; Patel et al., 2007), but in the beetle Tribolium (Patel et al., 2007), as well as in the myriapod Glomeris, the tips of outgrowing limbs do not express disco, and in the onychophoran expression is weaker in the distal region (compared to the median region of the legs) (summarized in Figure 5), implying a special function in distal limb development in Panarthropoda. ACKNOWLEDGMENTS Gene cloning and whole mount in situ hybridization experiments were conducted during the Evolution and Development course at Uppsala University in spring 2014. I wish to thank all students of this course for their contribution. Giannis Kesidis is thanked for his help as laboratory assistant. Nico Posnien and Alistair McGregor helped with the analysis of the embryonic transcriptomes. 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